Method for rapidly blocking spread of antibiotic resistance genes in water

Abstract

This application discloses a method for rapidly blocking the spread of antibiotic resistance genes (ARGs) in water. Addressing the shortcomings of existing single-radical oxidation disinfection technologies or the disordered combination of singlet oxygen and strong oxidizing free radicals in efficiently and cost-effectively degrading antibiotic resistance genes (ARGs) in water, this application proposes a sequential relay oxidation strategy. This strategy involves first using a certain concentration of singlet oxygen or Mn(III) to gently disrupt bacterial cell walls, followed by a lower concentration of free radicals to deeply degrade the resistance genes. This significantly inhibits the oxidative stress response of bacteria during disinfection, overcoming the technical problem of existing disinfection methods' inability to efficiently degrade intracellular ARGs.

Description

Technical Field

[0001] This application relates to the field of advanced oxidation water treatment technology, specifically to a method for rapidly blocking the spread of antibiotic resistance genes in water. It describes a method for efficiently degrading antibiotic resistance genes in water using a relay of singlet oxygen and strong oxidizing free radicals in complex water quality conditions. The method initiates / enhances the reaction through activation or external energy input, sequentially constructing a two-stage synergistic combined oxidation system of "singlet oxygen non-free radical oxidation cell disruption - strong free radical oxidation gene degradation," thereby efficiently removing antibiotic resistance genes from water. Background Technology

[0002] Antibiotic resistance genes (ARGs) are an emerging environmental pollutant. Because they can be spread between different bacteria through horizontal gene transfer (HGT), they continue to exist and spread even in the absence of antibiotic selection pressure, posing a serious threat to public health and ecological security.

[0003] Conventional wastewater treatment processes (such as sedimentation and activated sludge processes) and disinfection technologies (such as chlorination and ultraviolet irradiation) can effectively remove conventional pollutants and some microorganisms, but their ability to remove trace and persistent ARGs in water is limited. Current research indicates that the treatment environment of activated sludge processes not only fails to completely destroy free or intracellular ARGs, but may also induce bacterial stress responses due to low-concentration antibiotic residues, significantly increasing the frequency of HGTs and unintentionally becoming a potential breeding ground for drug-resistant bacteria accumulation and ARG amplification. Upgraded technologies such as membrane bioreactors (MBRs) perform excellently in solid-liquid separation, but their core problems are: membrane fouling leading to increased energy consumption and maintenance costs; simultaneously, ultrafiltration / microfiltration membranes cannot retain nanoscale free ARG fragments, and biodegradation also has limited effectiveness in degrading stubborn ARGs, instead leading to ARG accumulation within the system or in residual sludge, posing a secondary environmental risk of concentration-transfer. Chemical oxidation methods (Fenton's reagent, ozone oxidation) rely on strongly acidic conditions or high-concentration oxidants, producing iron sludge byproducts or high energy consumption problems. While chlorine disinfection is widely used, the required chlorine dose (>60 mg Cl2·min / L) for effectively inactivating drug-resistant bacteria is impractical in real-world applications. Furthermore, chlorination alters the microbial community structure, screens for chlorine-resistant bacteria, and induces increased cell membrane permeability, promoting conjugation and translocation. Ultraviolet disinfection, on the other hand, requires extremely high irradiation intensity (>160 mJ / cm²). 2 Only when these steps are taken can ARGs be significantly damaged, far exceeding the actual operating capacity of water plants. Photocatalytic disinfection may cause bacteria to enter a sub-lethal state, posing a risk of dark remediation and regeneration after treatment. Summary of the Invention

[0004] To address the aforementioned technical problems, this application provides a method for rapidly blocking the spread of antibiotic resistance genes in water. In complex water quality environments, this method efficiently degrades antibiotic resistance genes in water through a time-series relay of singlet oxygen and strong oxidative free radicals. While avoiding triggering bacterial oxidative stress responses, this method achieves a synergistic removal effect of "gentle cell disruption and efficient chain breaking," significantly improving the removal efficiency of resistance genes and the utilization efficiency of oxidants. This solves the problem of low efficiency and difficulty in achieving stable and efficient removal of antibiotic-resistant bacteria and their resistance genes in complex and variable solid water bodies using existing technologies.

[0005] Therefore, this application provides a method for rapidly blocking the spread of antibiotic resistance genes in water, the method comprising the following stages:

[0006] In the first stage, wastewater containing antibiotic-resistant bacteria and their resistance genes is treated to form non-free radical species in the wastewater. These non-free radical species are then used to oxidize and disrupt the cell membrane structure of the antibiotic-resistant bacteria, exposing their internal resistance genes to the oxidation system. The non-free radical species are singlet oxygen or Mn(III)aq.

[0007] In the second stage, a free radical-dominated oxidation system reaction is generated in the wastewater treated in the first stage to break down and mineralize the exposed drug resistance genes, thereby blocking their spread in the aquatic environment. In the second stage, the free radicals are selected from one or more of the following: hydroxyl radicals (•OH), superoxide radicals (O₂). 2•− iodate free radicals (IO) 3• Chlorine-active species.

[0008] Preferably, in the first stage, wastewater containing antibiotic-resistant bacteria and their resistance genes is treated to generate singlet oxygen in the wastewater. 1 O2 is maintained for 1 to 5 minutes, and singlet oxygen in the wastewater is maintained. 1 The steady-state concentration of O2 remains at 1×10⁻⁶. -10 mol / L or higher; or generate Mn(III)aq, maintaining the steady-state concentration of Mn(III)aq at 10⁻ 7 mol / L~10⁻ 5 mol / L; meanwhile, in the first stage, the steady-state concentration of each free radical remained at 1×10 mol / L. -13 Below mol / L.

[0009] Preferably, in the second stage, the reaction solution obtained after the first stage is further treated to allow the free radicals generated in the reaction solution to continue processing for 5 to 15 minutes; wherein, the steady-state concentration of each free radical is maintained at 1 × 10⁻⁶. -12 Above mol / L.

[0010] Preferably, the chlorine-active species CRS is Cl•, ClO•, and Cl2•. - One or more of them.

[0011] Preferably, the first and second stages are implemented by the following electrochemical methods to promote the generation of singlet oxygen and free radicals:

[0012] (1) Add an electrolyte to the wastewater. The electrolyte is selected from one or more of hydrochloride or sulfate, and the electrolyte is selected from at least one of NaCl, KCl, Na2SO4, and K2SO4. The concentration of the electrolyte is 40 mmol / L to 150 mmol / L.

[0013] (2) The current density is controlled at 2~30 mA / cm. 2 The optimal current density is 12.55 mA / cm². 2 ;

[0014] (3) First stage: Add the first oxidant, which is selected from at least one of hydrogen peroxide, sodium hypochlorite, persulfate monosalt (PMS), and potassium permanganate (PM); the dosage of the first oxidant in the wastewater is 0.01 g / L to 1 g / L; adjust the pH of the reaction system to 7 to 9 by adding alkali or by the reactor itself.

[0015] (4) Second stage: Add a second oxidant, which is selected from peracetic acid or periodate; the dosage of the second oxidant in the wastewater is 0.01 g / L to 1 g / L.

[0016] Preferably, the wastewater also includes at least one interfering substance, which is selected from at least one of dissolved organic matter, humic acid, bicarbonate ions, chloride ions, and hydrogen phosphate ions; and in the presence of the interfering substance, the method has an inactivation rate of >6.56 log for antibiotic-resistant bacteria.

[0017] Compared with the prior art, this application has the following beneficial effects:

[0018] 1. This application addresses the technical problems of existing single-radical oxidation easily triggering bacterial oxidative stress defense, leading to the difficulty in degrading intracellular antibiotic resistance genes; the insufficient capacity of single non-radical oxidation to completely mineralize resistance genes; and the easy quenching and unstable removal efficiency of multiple reactive oxygen species under complex water conditions due to disordered combinations. It constructs a two-stage sequential relay oxidation system of "singlet oxygen non-radical oxidation cell disruption—strong free radical oxidation gene degradation," achieving precise synergy between "cell disruption" and "chain breaking." First, it utilizes the selective oxidation of singlet oxygen or Mn(III)... The gentle oxidation process disrupts the bacterial cell membrane structure, avoiding the oxidative stress response caused by direct attack from strong free radicals. It significantly inhibits the secretion of extracellular polymers and intracellular antioxidants, removing the bioprotective barrier of intracellular antibiotic resistance genes and creating direct and sufficient contact conditions for subsequent efficient degradation by free radicals. Then, the exposed resistance gene fragments are efficiently degraded by strong free radicals through chain breaking and mineralization. Under optimal conditions, the average degradation efficiency of 19 antibiotic resistance genes reaches over 99.3% within 10 minutes, with an average removal log of 2.79 log copies / mL. The inactivation rate of antibiotic-resistant bacteria is greater than 6.56 log, effectively solving the technical problem that existing single oxidation technologies are unable to efficiently and thoroughly remove intracellular antibiotic resistance genes.

[0019] 2. This application introduces a non-radical pathway of singlet oxygen or Mn(III) with strong anti-interference ability in the critical early stage. It is not easily quenched by background components such as dissolved organic matter, bicarbonate, and chloride ions in the water, so that the system can maintain stable treatment performance in a wide range of pH 3-11 and complex water quality background. At the same time, singlet oxygen or Mn(III) preferentially acts on the microbial interface, which greatly reduces the ineffective reaction between active species and the background matrix of the water, and significantly improves the selectivity of the oxidation process for target bacteria and antibiotic resistance genes. The stable cell wall disruption effect in the early stage further ensures the target specificity of the subsequent free radical degradation stage. The two interact to solve the problems of severe loss of active species and large fluctuation of removal efficiency in traditional free radical oxidation systems under complex water quality.

[0020] 3. The two-stage sequential oxidation system constructed in this application has the characteristics of high selectivity and low ineffective loss. This allows the application to significantly reduce the total dosage of oxidant and improve the utilization efficiency of oxidant while achieving or even surpassing the treatment effect of traditional advanced oxidation processes. This, in turn, reduces the operating cost of water treatment. At the same time, the orderly design of the pathway avoids excessive reaction between high concentrations of oxidant and halide ions in the water, reduces the risk of generating toxic halogenated disinfection byproducts, improves the environmental friendliness of the process, and solves the problem of high cost and high risk of secondary pollution caused by the need to add excessive oxidant in order to pursue treatment effect in traditional methods.

[0021] 4. This application adopts an electrochemical treatment method, which achieves a universal control strategy for precise and controllable switching of the two-stage oxidation pathway by adjusting the reaction conditions such as the oxidant addition node, the type and activation method of the oxidant, pH value, and current density. It does not require large-scale modification of existing water treatment facilities and is easy to couple and upgrade with municipal sewage, hospital wastewater, and aquaculture wastewater treatment systems. It provides a reliable and promising technology option for the in-depth control of antibiotic resistance in different scenarios. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a graph showing the changes in spin-trapping EPR spectra recorded at different reaction times during the EF / sodium hypochlorite / hydrogen peroxide reaction.

[0024] Figure 2 The graphs show the inactivation curves of antibiotic-resistant bacteria in different systems of Examples 1 and Comparative Examples 2-5 of this application.

[0025] Figure 3 The graphs show the degradation performance of 19 resistance genes (ARGs) in different systems of Examples 1, 3, 4 and 5 of this application.

[0026] Figure 4 This is a graph showing the amount of EPS generated in MDR Escherichia coli in different systems of Examples 1, 3, 4, and 5 of this application.

[0027] Figure 5 The CT value and specific energy consumption of ARG degradation in Examples 1, 3, and 4 of this application and in previously reported advanced disinfection processes are evaluated.

[0028] Figure 6 The graphs show the inactivation curves of different systems against resistant bacteria in Examples 2 and Comparative Examples 6-11 of this application.

[0029] Figure 7 The degradation kinetics curves of pollutants in the EF / PM system after adding different active species quenchers in Example 2 of this application are shown.

[0030] Figure 8 The images show the UV-Vis absorption spectra at different time points during the reaction of the EF / PM system in Example 2 of this application.

[0031] Figure 9 This is an electrochemical reactor and schematic diagram of Example 3 of this application.

[0032] Figure 10 The above are EPR spectra of the EF / PMS system under different spin trapping agents (DMPO and TEMP) during the reaction process in Example 3 of this application.

[0033] Figure 11 The degradation kinetics curves of pollutants in the EF / PMS system after adding different active species quenchers in Example 3 of this application are shown. Detailed Implementation

[0034] The technical solutions of this application will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. All other embodiments obtained by those skilled in the art based on this application are within the scope of protection of this application. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments used are not specified, they can be obtained commercially.

[0035] Unless otherwise specified in the specific circumstances, the numerical ranges listed herein include upper and lower limits, as well as all integers and fractions within those ranges, and are not limited to the specific values ​​listed when defining the range. For numerical ranges, endpoint values ​​of various ranges, endpoint values ​​of various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. The term "and / or" used herein is inclusive; for example, "A and / or B" means only A, or only B, or both A and B. Terms such as "about," "substantially the same," and similar expressions herein indicate that reasonable deviations are permissible.

[0036] I. A method for rapidly blocking the spread of antibiotic resistance genes in water

[0037] This application addresses the technical problems in existing technologies, such as the difficulty in degrading intracellular antibiotic resistance genes due to the easy triggering of bacterial oxidative stress defense by single free radical oxidation disinfection, the inability of single non-free radical oxidation capacity to completely mineralize resistance genes, and the inability of disordered combination of multiple reactive oxygen species to resolve the above contradictions, as well as the easy quenching of reactive species and unstable removal efficiency under complex water conditions. Based on the understanding that the bacterial cell membrane is the main protective barrier for intracellular resistance genes and that there are significant differences in the oxidative selectivity and oxidative capacity of different reactive oxygen species, this application proposes a technical approach to temporally regulate the composition and generation sequence of reactive species in the oxidation system. First, a non-free radical oxidation system dominated by singlet oxygen or Mn(III) is constructed to selectively destroy the bacterial cell membrane structure. Then, an oxidation system dominated by strong oxidative free radicals is constructed to degrade exposed resistance genes. By constructing oxidation systems with different dominant reactive species in stages, the problem of a single oxidation pathway is avoided. Furthermore, by adjusting the oxidant addition node, oxidant type and activation method, pH value, current density, temperature, light intensity, and other reaction conditions, the precise and controllable switching of the two-stage oxidation pathway can be achieved to adapt to the complex and variable actual water environment. Therefore, the method designed in this application is carried out in the following stages:

[0038] In the first stage, wastewater containing antibiotic-resistant bacteria and their resistance genes is treated to form non-free radical species in the wastewater. These non-free radical species are then used to oxidize and disrupt the cell membrane structure of the antibiotic-resistant bacteria, exposing their internal resistance genes to the oxidation system. The non-free radical species are singlet oxygen or Mn(III)aq.

[0039] In the second stage, a free radical-dominated oxidation system reaction is generated in the wastewater treated in the first stage to break down and mineralize the exposed drug resistance genes, thereby blocking their spread in the aquatic environment. In the second stage, the free radicals are selected from one or more of the following: hydroxyl radicals (•OH), superoxide radicals (O₂). 2•− iodate free radicals (IO) 3• Chlorine-active species.

[0040] The two-stage sequential oxidation system designed in this application has achieved many unexpected technical effects in practical applications: the first stage, dominated by singlet oxygen, can complete the perforation and disruption of bacterial cell membranes within only 1 to 5 minutes, and the amount of extracellular polymers secreted by bacteria during this process is reduced by about 32% compared to the single chlorine radical oxidation system, effectively avoiding the obstruction of subsequent degradation processes by bacterial defense mechanisms. Based on this, the strong oxidizing free radicals in the second stage can directly act on drug resistance genes released from the cell, achieving an average degradation efficiency of over 99.3% for 19 antibiotic resistance genes within 10 minutes, with an average removal log value of 2.79 log copies / mL. In contrast, under the same conditions, the average removal log values ​​for drug resistance genes by the single electroactivated sodium hypochlorite system and the single electroactivated hydrogen peroxide system are only 0.453 log copies / mL and 0.600 log copies / mL, respectively. Meanwhile, even in complex water conditions containing high concentrations of dissolved organic matter, bicarbonate ions (up to 161 mg / L), and chloride ions (up to 90.8 mg / L), the method described in this application achieves an inactivation rate of greater than 6.56 log for antibiotic-resistant bacteria across a wide pH range of 3–11, while maintaining a removal rate of over 99% for resistance genes. In contrast, traditional free radical oxidation systems show a significant decrease in treatment efficiency under the same interference conditions. Furthermore, the specific energy consumption required for the method described in this application to degrade 1-log antibiotic resistance genes is only 0.06 kWh·m³. -3 ・log -1 The required CT value is 28 to 53 times lower than other advanced oxidation processes based on the combination of sodium hypochlorite and hydrogen peroxide, which significantly improves processing efficiency while greatly reducing operating costs and oxidant consumption.

[0041] In some embodiments of this application, in the first stage, wastewater containing antibiotic-resistant bacteria and their resistance genes is treated to generate singlet oxygen in the wastewater. 1 O2 and maintain treatment time of 1 min to 5 min, and singlet oxygen in wastewater 1 The steady-state concentration of O2 remains at 1×10⁻⁶. -10 The concentration of free radicals in the first stage is above 1 mol / L; meanwhile, the steady-state concentration of free radicals in the first stage remains at 1 × 10⁻⁶. -13The concentration is below mol / L. This application is designed to address the problems of disordered composition of active species in existing oxidation systems and the tendency of strong free radicals to prematurely trigger bacterial oxidative stress defenses. Existing technologies typically employ strong free radical oxidation systems or mixed systems of free and non-free radicals when treating intracellular resistance genes. Strong oxidizing free radicals come into contact with bacteria during the cell disruption stage, inducing the bacteria to initiate oxidative stress pathways, promoting extracellular polymer secretion and intracellular antioxidant enzyme synthesis, forming a dual physical and chemical protective barrier on the bacterial surface, hindering the penetration of oxidizing species into the cell, ultimately making it difficult to effectively degrade intracellular resistance genes. This application, by limiting the lower limit of the steady-state concentration of singlet oxygen in the first stage and the reaction time, ensures sufficient reaction kinetics for non-free radical oxidation, selectively oxidizing and destroying the phospholipid bilayer structure of the bacterial cell membrane within a short time of 1-5 minutes, rapidly achieving cell membrane perforation and the release of intracellular resistance genes, ensuring the timeliness and sufficiency of the cell disruption process; simultaneously, it strictly controls the steady-state concentration of free radicals in the first stage at 1×10⁻⁶. - 13 At concentrations below mol / L, strong free radicals can be completely avoided from intervening in the cell disruption stage, preventing bacteria from activating their defense mechanisms after sensing oxidative stress and effectively reducing the generation of extracellular polymers and the synthesis of intracellular antioxidants. These two aspects also exhibit a significant synergistic effect: a sufficient concentration of singlet oxygen provides stable and efficient cell disruption capability, while the extremely low free radical base concentration eliminates the trigger for bacterial defense mechanism activation. Under their combined action, cell membrane structure destruction can be completed in a short time without inducing a protective response in bacteria. This allows intracellular drug resistance genes to be fully and unprotectedly exposed to the reaction system, providing direct targets for the second-stage free radical degradation. Simultaneously, it reduces the generation of antioxidant interference substances in the system, lowers the subsequent ineffective loss of free radicals, and improves the overall efficiency of the oxidation system.

[0042] In some embodiments of this application, in the first stage, wastewater containing antibiotic-resistant bacteria and their resistance genes is treated to maintain a steady-state concentration of Mn(III)aq in the wastewater at 10. -7 mol / L~10 -5 mol / L; meanwhile, the steady-state concentration of free radicals in the first stage remained at 1×10 mol / L. -13Below mol / L. This design addresses the problem in existing manganese-based oxidation processes where the valence state of active species is uncontrollable and easily leads to the generation of large amounts of free radicals, thus triggering bacterial oxidative stress. Existing technologies using manganese-based reagents to treat wastewater typically directly generate high-valence manganese species or trigger free radical chain reactions. Their strong oxidizing effect induces bacterial stress defense in the early stages of contact with bacteria. Furthermore, when the concentration of Mn(III), as an intermediate valence species, is not effectively controlled, either the concentration is too low, resulting in insufficient oxidative cell disruption, or the concentration is too high, leading to over-oxidation and conversion to higher valence states, failing to achieve a gentle and selective cell disruption effect. This application limits the steady-state concentration of water-soluble Mn(III) to 10 mol / L. -7 ~10 -5 Within a suitable range of mol / L, it can utilize the moderate oxidation potential and interfacial reaction characteristics of Mn(III) to selectively oxidize functional groups on the bacterial cell membrane surface, thereby disrupting the cell membrane's integrity and selective permeability, and efficiently releasing intracellular resistance genes. Its mild oxidative intensity will not directly trigger a severe oxidative stress response in bacteria; simultaneously, combined with 1×10⁻⁶ mol / L... - 13 Controlling the free radical concentration below mol / L ensures that the first-stage oxidation process is entirely dominated by Mn(III), preventing free radicals from prematurely attacking the bacterial cells and disrupting the selectivity and gentleness of the cell disruption process. This concentration range of Mn(III) synergistically complements the low free radical baseline conditions. A suitable concentration of Mn(III) provides stable and controllable cell disruption kinetics, while the strictly limited free radical concentration ensures high selectivity in the oxidation process. This synergy allows for effective disruption of the cell membrane structure without activating the bacterial antioxidant defense system, while preventing excessive conversion of Mn(III) into highly oxidizing, high-valence species or triggering free radical side reactions. This ensures the controllability of the cell disruption process and reduces the generation of interfering substances such as extracellular polymers, providing a low-interference reaction environment for subsequent free radical degradation stages and further enhancing the efficiency of subsequent gene chain fragmentation degradation.

[0043] In some embodiments of this application, in the second stage, the reaction solution obtained after the first stage treatment is further treated to generate second-stage free radicals, and the treatment continues for 5 to 15 minutes; wherein, the second-stage free radicals are selected from one or more of the following free radicals: hydroxyl radical •OH, iodate radical IO3. • Superoxide radicals (O2•) - Sulfate free radicals SO4• - Chlorine-reactive species CRS; the steady-state concentration of each second-stage free radical remains at 1×10⁻⁶. -12The concentration is above mol / L. This stage is designed to address the problems in existing technologies, such as unclear free radical oxidation targets, susceptibility to quenching by biological barriers and intracellular defense substances, and low efficiency in degrading intracellular drug-resistant genes. Existing technologies typically generate strong oxidizing free radicals directly in systems containing intact bacteria. These free radicals need to penetrate the bacterial cell membrane and extracellular polymeric barriers to reach nucleic acid molecules, and are extensively quenched by intracellular antioxidant enzymes and reducing substances during this process. This results in extremely low free radical utilization efficiency, requiring very high oxidant dosages and longer reaction times to achieve limited gene degradation, making it impossible to balance processing efficiency and operating costs. This application initiates the free radical oxidation stage after the first stage of cell disruption, and controls the steady-state concentration of each free radical at 1×10⁻⁶. -12 At concentrations above mol / L, under the premise of sufficient exposure of drug resistance genes, the high oxidation potential of strong oxidizing free radicals can continuously attack the phosphodiester bonds and base structures of nucleic acid molecules, causing chain breakage and mineralization of drug resistance genes, completely losing their biological activity and horizontal transfer ability. The reaction time of 5-15 minutes can ensure sufficient degradation. At the same time, since the first stage has removed the biological protective barrier of bacteria and inhibited the secretion of defense substances, free radicals do not need to penetrate biological structures and will not be quenched by a large number of intracellular antioxidants. Their target is more specific, and the oxidation utilization efficiency is significantly improved. There is a close temporal synergistic effect between the concentration of free radicals and the pretreatment of cell disruption. Sufficient concentration of free radicals provides strong gene degradation ability, while the barrier-free reaction environment constructed by the first-stage pretreatment allows the oxidative efficiency of free radicals to be fully released. The two form a temporal relay of "cell disruption-chain breaking", which not only avoids the ineffective loss and negative stress-induced effect of free radicals in the cell disruption stage, but also concentrates on exerting strong oxidative ability in the gene degradation stage. This significantly shortens the reaction time and improves the degradation depth of drug resistance genes, achieving a complete transmission blocking effect from cell inactivation to gene mineralization. This effectively solves the technical problem of low efficiency in degrading intracellular ARGs by single free radical process.

[0044] In some embodiments of this application, the chlorine-active species CRS is Cl•, ClO•, and Cl2•. -One or more of these. When using chlorine disinfection to treat wastewater containing ARGs, existing technologies mainly use free chlorine as the active species. Its ability to degrade nucleic acids is limited, making it difficult to completely destroy the molecular structure of drug-resistant genes. Moreover, in order to effectively inactivate drug-resistant bacteria, high doses of chlorine preparations need to be added. High concentrations of free chlorine will react with natural organic matter in the water to produce a large amount of toxic disinfection byproducts such as trihalomethanes and haloacetic acids, which bring secondary environmental risks. At the same time, the composition of active species in traditional chlorine disinfection is uncontrollable and cannot adapt to the differentiated functional requirements of staged cell wall disruption and chain breaking. This application explicitly uses chlorine-based free radicals as the active species in the second stage. These chlorine-based free radicals have a higher redox potential than free chlorine, which can effectively attack the sugar rings and base structures of nucleic acid molecules and break phosphodiester bonds, thereby achieving efficient degradation and bioactivation of drug resistance genes. Their gene degradation ability is significantly better than that of traditional free chlorine disinfection. At the same time, these chlorine-based free radicals are generated on demand in the second stage, avoiding the long-term retention of high concentrations of free chlorine in the system, reducing the time and probability of halogenation side reactions with background organic matter in the water, and reducing the potential for the formation of toxic halogenated disinfection byproducts. The strong degradation ability of chlorine-based free radicals and their sequential generation mode create a synergistic effect. The high oxidation potential of chlorine-active free radicals ensures the degradation depth and complete inactivation of drug-resistant genes, while the phased generation mode solves the byproduct problem caused by the long-term high-chlorine environment of traditional chlorine disinfection. At the same time, combined with the cell wall disruption effect of the first stage, chlorine-active free radicals can directly act on exposed nucleic acid targets without breaking through the bacterial biological barrier, further reducing the amount of chlorine-based oxidant required to achieve the same degradation effect. This not only improves the treatment efficiency but also enhances the environmental safety of the process, achieving a balanced optimization of treatment effect and ecological risk.

[0045] In some embodiments of this application, the wastewater also includes at least one interfering substance selected from at least one of dissolved organic matter, humic acid, bicarbonate ions, chloride ions, and hydrogen phosphate ions; and in the presence of the interfering substance, the method achieves an inactivation rate of >6.56 log for antibiotic-resistant bacteria. In existing free radical oxidation processes treating actual wastewater, the widely present dissolved organic matter, humic acid, and various anions and cations in the water act as free radical quenchers, consuming large amounts of active oxides, leading to a significant decrease in oxidation efficiency. The treatment effect fluctuates significantly with water quality, making it difficult to consistently achieve the expected inactivation of antibiotic-resistant bacteria and removal of ARGs. Furthermore, in engineering practice, to offset matrix interference, the dosage of oxidant is typically increased significantly, further increasing operating costs and exacerbating the risk of byproduct formation. In the two-stage oxidation system constructed in this application, the first stage, which is a non-radical oxidation pathway dominated by singlet oxygen or Mn(III), has low sensitivity to the aforementioned common water matrix and is not easily quenched. It can stably exert its bacterial cell membrane destruction effect in an environment where interfering substances coexist, ensuring the stability of the inactivation efficiency of drug-resistant bacteria. At the same time, since the first stage has completed the cell wall disruption and reduced the generation of extracellular polymers, the free radicals in the second stage can act on the exposed drug-resistant genes in a targeted manner, reducing the probability of ineffective collision reactions with the background water matrix. Thus, it can still maintain a stable gene degradation efficiency under matrix interference. The anti-interference characteristics of the first stage and the targeted action of the second stage form a supporting synergistic effect. The strong resistance to matrix interference of the non-radical pathway in the first stage provides a stable foundation for cell disruption in the entire treatment process, ensuring that the bacterial cell structure can be effectively destroyed under complex water quality. The targeted degradation of free radicals in the second stage further reduces the dilution effect of the background matrix on the oxidation efficiency. The two stages are connected and mutually supportive, enabling the system to maintain a drug-resistant bacteria inactivation rate of >6.56 log and a high efficiency of ARGs degradation under complex water quality conditions containing multiple interfering substances. This solves the technical problems of poor resistance to water quality fluctuations and unstable treatment effects of traditional free radical oxidation processes, and significantly improves the engineering applicability of the process in actual wastewater treatment scenarios.

[0046] In some embodiments of this application, in the first and second stages, the process of preparing singlet oxygen or free radicals by various means in the prior art is not difficult to achieve. The focus of this application is not on what means to prepare singlet oxygen or free radicals, but on controlling singlet oxygen and free radicals within the range at different stages so that they can play corresponding roles at different stages, thereby degrading antibiotic resistance genes generated by bacterial inactivation while inactivating antibiotic-resistant bacteria, and blocking their spread in the aquatic environment.

[0047] In some embodiments of this application, in the first or second stage, the reaction is induced by applying activation to the wastewater or inputting external energy, thereby generating the singlet oxygen or free radicals. The application of activation or input of external energy is selected from methods that preferentially generate singlet oxygen or free radicals through electrochemical means.

[0048] In some embodiments of this application, singlet oxygen is generated through electrochemical activation in the first or second stage. In this process, the electrolyte is one or more of a hydrochloride or sulfate; the sulfate is at least one of sodium sulfate or potassium sulfate; the hydrochloride is at least one of sodium chloride and potassium chloride; the concentration of the electrolyte is 40 mmol / L to 150 mmol / L; and the current density of the electric field treatment is 2 mA / cm². 2 ~30 mA / cm 2 The anode of the chemical system electrode is selected from graphite electrodes, metal electrodes, or metal composite electrodes. Graphite electrodes include graphite rod electrodes, graphite filament electrodes, graphite felt electrodes, graphite plate electrodes, graphite sponge electrodes, graphite particle electrodes, or porous graphite electrodes. The cathode is selected from carbon-based material electrodes, titanium material electrodes, stainless steel electrodes, or metal composite electrodes. Preferably, it is a carbon-based material electrode with high specific surface area, abundant pore structure, and excellent conductivity to provide a large number of active sites and significantly improve reaction activity. The metal composite electrode refers to a composite electrode based on metal elements from periods 4 to 6 of the periodic table and modified by one or more of metals, metal oxides, or metal hydroxides, including but not limited to titanium-plated platinum electrodes or nickel-plated platinum electrodes. This electrode can be applied to the anode or cathode side according to system design requirements to achieve specific electrocatalytic performance and operational stability.

[0049] In some embodiments of this application, a first oxidant is added in the first stage. The first oxidant is selected from at least one of hydrogen peroxide, sodium hypochlorite, monosodium persulfate, and potassium permanganate. The dosage of the first oxidant in the wastewater is 0.01 g / L to 1 g / L. The pH of the reaction system is adjusted to 7 to 9. A second oxidant is added in the second stage. The second oxidant is selected from peracetic acid or periodate. The dosage of the second oxidant in the wastewater is 0.01 g / L to 1 g / L. The first oxidant is selectively added in the first stage of the reaction to preferentially generate an oxidation system dominated by singlet oxygen or Mn(III)aq non-radical active species. The second oxidant is added in the second stage of the reaction. At this time, since the bacterial cells have been destroyed, a large amount of cell contents are released into the water. The wastewater after the first stage treatment generates a free radical-dominated oxidation system reaction, which breaks down and mineralizes the exposed drug resistance genes, blocking their spread in the aquatic environment, thereby achieving deep degradation and mineralization of dissolved organic matter. In the second stage, the free radical is selected from one or more of the following: hydroxyl radical •OH, superoxide radical O2. •− iodate free radical IO3 • Chlorine-active species.

[0050] In some embodiments of this application, to illustrate how the two stages described in this application are carried out, the E / H2O2 / NaClO system is used as an example for explanation, but it is not limited to this system, as follows:

[0051] Phase 1: 1 O2 generation

[0052] H₂O₂ + HClO → 1 O2 + H2O + H + +Cl -

[0053] H2O2+OH - →HO2 - +H2O

[0054] HO2 - +HClO→ 1 O2 + H2O + Cl -

[0055] Second stage: After the oxidant (H2O2) is depleted, chlorine free radicals dominate the generation:

[0056] HClO→H + +ClO -

[0057] ClO - +e - →ClO •-

[0058] HClO+e - →Cl•+OH -

[0059] Cl•+Cl - ⇌ Cl2 •-

[0060] Cl• + HClO → ClO• + Cl - +H +

[0061] The above reaction is an illustrative explanation of the method described in this application. In the first stage, the first oxidant added can be selected from one or more of hydrogen peroxide / sodium hypochlorite, single persulfate (PMS), and potassium permanganate (PM) to preferentially generate singlet oxygen rather than free radical reactive species. In the second stage, the second oxidant added can be selected from one or more of periodate and peracetic acid oxygen to initiate and enhance free radical generation. However, it should be noted that the second stage may also not require the addition of an oxidant or peroxidant, such as EF / hydrogen peroxide / sodium hypochlorite. When the hydrogen peroxide is consumed, the system spontaneously transforms into a system that generates chlorine free radicals.

[0062] In some embodiments of this application, under the sequential oxidation of singlet oxygen and free radicals described above, the average degradation efficiency of 19 antibiotic resistance genes (ARGs) reached over 99.3% within 10 minutes (average log removal value of 2.79 log copies / mL).

[0063] It is evident that there is a significant synergistic effect between non-radicals and free radicals. The singlet oxygen non-radical oxidation-cell disruption-free radical strong oxidative degradation gene-coupled oxidation system constructed in this application first utilizes… 1 O2 rapidly disrupts cell membrane structure, causing perforation and releasing intracellular resistance genes within 1-5 minutes. Subsequently, various strong oxidizing free radicals efficiently degrade extracellular resistance genes within 5-15 minutes. This coupled system demonstrates a significantly higher removal efficiency of antibiotic resistance genes in water compared to single free radical systems, fully showcasing the synergistic advantages of relay oxidation by non-free radicals and free radicals.

[0064] II. Examples and Comparative Examples

[0065] Example 1

[0066] In this embodiment: the initial concentration of multidrug-resistant Escherichia coli in the wastewater was 10. 7 The reactor has a capacity of CFU / mL and is a cylindrical flow-through electrochemical reactor with an internal chamber. The reactor has an outer radius of 10 cm and a thickness of 4.8 cm, and the internal chamber has a diameter of 4 cm and a depth of 1.5 cm.

[0067] The specific steps of this embodiment include: thoroughly stirring the wastewater at 800 r / min using a magnetic stirrer, adding sodium sulfate to achieve a concentration of 50 mmol / L, then sequentially adding sodium hypochlorite and hydrogen peroxide solution to achieve a sodium hypochlorite concentration of 0.1 mg / L and a hydrogen peroxide concentration of 0.5 mmol / L. The prepared wastewater is then injected into a flow-through electrochemical reactor, using activated carbon fiber as the cathode and a titanium-plated platinum electrode as the anode, at a speed of 6.41 mA / cm². 2 The electrolysis reaction was initiated at a current density of [value missing]. During the process, 1 mL samples were taken at 0, 2, 4, 6, 8, and 10 min, and immediately quenched with sodium thiosulfate. The samples were then subjected to [further treatment / processing]. 1 Up to 10 5 After serial dilution, take 0.1 mL and dilute 10 times. 5 A solution diluted 10 times was spread onto agar plates containing tetracycline (16 mg / L), incubated at 37°C for 24 h, and colony counts were performed to calculate the removal rate. DNA was extracted from the samples, and the copy numbers of 19 target resistance genes were detected by real-time quantitative PCR to assess the degradation effect. The system utilizes electro-induced pH stratification, preferentially generating singlet oxygen (SO4) in the alkaline microenvironment during the early stage of the reaction (0–2 min). 1 O2 selectively disrupts cell membranes and induces gene release; after hydrogen peroxide is consumed, the system spontaneously transitions to a chlorine radical-dominated phase (after 2 minutes), rapidly degrading exposed gene fragments, such as... Figure 1 As shown. The results indicate that complete bacterial inactivation (greater than 7.33 log) can be achieved within 10 min of reaction, as shown. Figure 2 As shown; the degradation capacity of the resistance gene reached 2.79 log copy number mL. -1 The removal rate is greater than 99.3%, such as Figure 3 As shown. The performance was stable over a wide pH range and in actual aqueous substrates, verifying the effectiveness of the "first disrupt the cell wall, then break the chain" relay oxidation strategy. In this embodiment, the bacterial removal rate was 4.92 log after 15 min. The obtained sample was centrifuged at 12,000 rpm to precipitate bacteria. The precipitate was washed with buffer to remove impurities. The precipitate was stirred at 45°C for 1 hour, centrifuged again to remove bacterial cell debris, and the supernatant was collected and aseptically filtered. The resulting solution was the EPS extract, and the total EPS content was measured to be 6.12 mg / L. When NaHCO3 (to scavenge chlorine free radicals) or NaN3 (to scavenge...) was added to the EF / NaClO / H2O2 system of this application... 1 After removing O2 and chlorine free radicals, the EPS content decreased to 1.3 and 1.15 mg / L, close to the background level. Specifically, as follows... Figure 4As shown. By analyzing the SEC value per unit energy consumption and the CT value of the concentration-time product, it was found that the specific energy consumption of this system is only 0.06 kWh·m³. -3 · log -1 The CT value required to achieve 1-log ARG degradation is extremely low; the CT requirement for EF / NaClO / H2O2 is 28-53 times lower than that of other NaClO / H2O2-based AOPs. Specifically... Figure 5 As shown. The effects of different hydrogen peroxide concentrations on the EF / sodium hypochlorite / hydrogen peroxide process. 1 The analysis of the influence of O2 steady-state concentration yielded the singlet oxygen content in wastewater. 1 The steady-state concentration of O2 remains at 1×10⁻⁶. -10 The concentrations are above mol / L, as shown in Table 1. The steady-state concentrations of active species in the EF / NaClO / H2O2 system of this invention are compared with those in other similar advanced oxidation processes. It is clearly stated that the steady-state concentration of chlorine-containing active species (CRS, i.e., free radicals) in this system remains at 1 × 10⁻⁶ mol / L. -12 mol / L and above, as shown in Table 2.

[0068] Table 1. Effect of hydrogen peroxide concentration on the EF / sodium hypochlorite / hydrogen peroxide process 1 Effect of O2 steady-state concentration

[0069]

[0070] Table 2. Comparison of oxidation mechanisms between the EF / sodium hypochlorite / hydrogen peroxide system and similar advanced oxidation processes.

[0071]

[0072] Example 2

[0073] In this embodiment, the initial concentration of ARE. coli in the wastewater was approximately 10. 7 CFU·mL -1 The experimental setup consists of a peristaltic pump, a cathode-flow electrochemical reactor, a 500 mL cylindrical glass beaker, and inlet and outlet water pipes. The reactor is a cathode-flow electrochemical reactor. The anode is a 39 mm diameter circular titanium-plated platinum electrode, and the cathode is a 39 mm diameter, 2 mm thick carbon fiber felt supported by a circular titanium mesh. It is powered by a conductive titanium foil, and the distance between the anode and cathode is 20 mm. A hole is opened in the reactor lid for detecting the pH value of the solution inside the reactor chamber.

[0074] The specific steps of this embodiment include: taking 1 mL of the prepared ARE. coli bacterial suspension, preparing 500 mL of experimental bacterial solution, adding it to a sterile glass beaker, and adding 50 mmol·L⁻¹ to the bacterial solution. -1Anhydrous sodium sulfate electrolyte was used, and the beaker was placed on a magnetic stirrer set to 800 rpm. The reaction temperature was 25 ℃ ± 1 ℃. The experimental water sample was thoroughly mixed. During the reaction, the experimental water sample was first flowed evenly through the reaction apparatus, with water entering at the anode and exiting at the cathode. When the experimental water sample began to flow through the cathode, the DC regulated power supply was immediately turned on and adjusted to the required current density of 12.55 mA·cm⁻¹. -2 Then, a concentration of 7 μmol·L⁻¹ was quickly added to the beaker. -1 Potassium permanganate solution was added, and timing was started. At preset sampling times of 0, 3, 5, 8, 10, and 15 minutes, 1 mL of the reactor cathode effluent was collected into centrifuge tubes containing 30 μL of 4% sodium thiosulfate solution. The samples were vortexed and mixed to quench the bacteria. The 1 mL of quenched bacterial culture was then diluted with 50 mmol·L⁻¹ water. -1 The sodium sulfate solution was serially diluted 10-fold to the appropriate concentration, and after thorough mixing, 100 μL was taken and evenly spread on LB agar medium (containing 16 μg·L⁻¹) using a sterile spreader. -1 Two plates were spread on each dilution gradient of tetracycline. The sterile plates were sealed and inverted in a 37°C incubator for 24 hours. Afterward, intact colonies were counted, and the bacterial removal rate was calculated. A time-logarithmic bacterial removal curve was plotted, as shown below. Figure 6 As shown. Simultaneously, to investigate the active species in this system, excess quenchers (such as methanol (MeOH), tert-butanol (TBA), L-histidine (L-His), dimethyl sulfoxide (DMSO), and PP) were added. The samples were vortexed and mixed to achieve quenching. By comparing the changes in pollutant degradation efficiency under different quenching conditions, the respective contributions of free radical and non-free radical pathways were clearly distinguished. The results are shown below. Figure 7 As shown. Using PMSO as a probe, the amount of PMSO2 generated was determined by HPLC-MS, and the transformation of manganese species during the reaction was monitored by UV-Vis spectroscopy, such as... Figure 8 As shown.

[0075] Example 3

[0076] In this example, the initial concentration of ARE. coli in the wastewater was approximately 10. 7 CFU·mL -1 The device is like Figure 9 As shown, a unidirectional electrofiltration device equipped with a platinum (Pt) plate anode and an activated carbon fiber (ACF) felt cathode was used. The single-pass electrofiltration reactor (acrylic material) had an electrode spacing of 1 cm and was equipped with a peristaltic pump and sampling port. The ACF cathode was soaked in 0.1 M HCl for 12 hours and rinsed with deionized water until neutral. The reactor tubing was rinsed with deionized water for 10 minutes. The final concentration of PMS added during the process was 150 μmol / L.

[0077] The specific steps of this embodiment include: taking 1 mL of the prepared ARE. coli bacterial suspension, preparing 500 mL of experimental bacterial solution, adding it to a sterile glass beaker, and adding 50 mmol·L⁻¹ to the bacterial solution. -1 Anhydrous sodium sulfate electrolyte was used, and the beaker was placed on a magnetic stirrer set to 800 rpm. The reaction temperature was 25 ℃ ± 1 ℃. The experimental water sample was thoroughly mixed. During the reaction, the experimental water sample was first flowed evenly through the reaction apparatus, with water entering at the anode and exiting at the cathode. When the experimental water sample began to flow through the cathode, the DC regulated power supply was immediately turned on and adjusted to the required current density of 12.55 mA·cm⁻¹. -2 Then, the PMS solution was pumped into the reactor at a flow rate of 5 mL / min, and timing was started. At preset sampling time points of 0, 3, 5, 8, 10, and 15 min, 1 mL of the reactor cathode effluent was collected in centrifuge tubes containing 30 μL of 4% sodium thiosulfate solution. To clarify the non-radical-dominated oxidation process and identify ¹O₂ as the core reactive species, EPR tests and quenching experiments were performed, such as... Figure 10 As shown. Further, different quenchers (such as methanol (MeOH), tert-butanol (TBA), L-histidine (L-His), and dimethyl sulfoxide (DMSO)) were added to inhibit specific active species. The contribution of each species was inferred by observing the degree of inhibition of pollutant degradation. The results are shown below. Figure 11 As shown.

[0078] To accurately track the scavenging effect of ¹O2 and Mn(III) hydrates on selected resistance genes (ARGs), this embodiment employs an optimized kinetic method, as shown in the formula below.

[0079]

[0080]

[0081] in, , , , , and This represents the damage kinetic constant (unit: min) of the selected resistance gene (ARG) during EF / PMS, PMS, EF / PMS / L-his, EF / PM, PM, and EF / PM / PP treatments. -1 ),Depend on Calculate, and Representing ARGs pairs 1The damage kinetic constants (unit: min⁻¹) of O₂ and Mn(III)aq are shown in Table 3. The actual steady-state concentration of this active species in the water body is calculated (unit: usually mol / L or M).

[0082] That is, Example 2:¹The steady-state concentration of O₂ is 7.0 × 10⁻⁶. -12 Above mol / L, Example 3: Mn(III) aq The steady-state concentration is 10 -7 mol / L to 10 -5 mol / L.

[0083] For ¹O2:

[0084] ;

[0085] For Mn(III) aq :

[0086] .

[0087] Table 3 Damage kinetic parameters of selected resistance genes (ARGs)

[0088]

[0089] Example 4

[0090] In this embodiment, a 250 mL circular glass beaker is used as the reactor. A thermostatic magnetic stirrer is placed below to stir the solution at 800 rpm while maintaining a stable reaction temperature. A 12.5 cm diameter acrylic circular cover is placed on the reactor, and an electrode clamp is fixed to the cover. An electrode plate is fixed to one side of the electrode clamp, and the other side is connected to the cathode and anode of a DC regulated power supply, respectively. The electrode plate is made of titanium platinum-plated (Ti / Pt) material, with a size of 3.5 × 5 cm and a cathode-cathode distance of 2 cm. Two additional holes are made on the cover plate: one for sampling and the other for detecting the pH value of the solution.

[0091] Take 0.5 mL of an E. coli suspension with the same initial concentration and add 100 mmol•L to the reactor. -1 The sodium sulfate electrolyte solution was used to make the reaction volume 200 mL, and the bacterial concentration in the reactor was approximately 10⁸ CFU / mL. -1 The pH of the solution was measured using a pH meter and 100 mmol·L⁻¹. -1 The pH was adjusted to a predetermined value using sulfuric acid solution and sodium hydroxide solution. During the reaction, the DC power supply was turned on first, followed by the addition of peracetic acid aqueous solution to bring the peracetic acid concentration in the reaction solution to 15 μmol•L. -1In the experiment, the current density is generally 60 mA•cm. -2 At reaction times of 0, 2, 4, 6, 8, and 10 min, 1 mL of reaction solution was taken and quenched with 50 μL of 4% sodium thiosulfate solution. The solution was then serially diluted in sterile water, and 100 μL samples were taken in triplicate. All experiments in this embodiment were repeated at least twice. The average results were calculated, and the standard deviation was plotted to generate error bars. The free radical quenching experiment demonstrated the existence of free radicals, and the contribution percentage of different free radicals was estimated. The results are shown in Table 4. It was further inferred that the steady-state concentration of each free radical remained at 1 × 10⁻⁶. -12 Above mol / L.

[0092] Table 4. Estimated contribution percentage of different oxidation pathways in the system

[0093]

[0094] In the table, A: anodizing; B: PAA inactivation; C: •OH inactivation; D: R–O• inactivation.

[0095] Example 5

[0096] The system includes a DC regulated power supply, a peristaltic pump, a flow-through cathode electrochemical reactor, a 500 mL cylindrical glass flask, and inlet pipes connected to each device. The reactor is topped with a 39 mm diameter circular titanium-plated anode, and its cathode is composed of a circular titanium mesh as the bottom material and a carbon fiber felt electrode with a thickness of 2 mm. The anode and cathode are spaced 20 mm apart and are connected to the power supply through conductive titanium foil.

[0097] For each test preparation, 1 mL of ARE. coli suspension was used to prepare 500 mL of test bacterial solution, with anhydrous sodium sulfate added to achieve an electrolyte concentration of 50 mmol·L⁻¹. –1 The glass beaker was placed on a magnetic stirrer at 800 rpm, and the experiment was conducted at room temperature. Before the reaction, the solution was injected into the reactor from both the anode inlet and the cathode outlet to ensure complete water permeation during the reaction. After the solution passed through the cathode, a DC regulated power supply was connected and adjusted to the set voltage. Sodium periodate was then poured into the beaker, and a timer was started. At predetermined sampling times (0, 2, 5, 8, 10, 12, and 15 min), 1 mL of solution was collected from the cathode end and quickly transferred to a centrifuge tube containing 50 µL of 4% sodium thiosulfate. The mixture was then vortexed and stirred until homogeneous before being stored at room temperature. Analysis of the obtained samples using EPR testing and masking agent experiments clearly revealed the presence of O2. •− ,1 O2, •OH, and IO3• are the main reasons why EC-PI inactivates ARE. coli within 15 min. The contribution percentages of different oxidation pathways in the system were calculated, and the results are shown in Table 5. From this, it can be deduced that the concentration of free radicals is around 1×10⁻⁶. -12 Above mol / L.

[0098] Table 5. Contribution rate of different oxidation pathways

[0099]

[0100] A: Carbon felt cathode electroadsorption; B: Active oxygen oxidation; C: 1 O2 pathway; D: caused by the synergistic effect of several pathways such as direct electron transfer.

[0101] Comparative Example 1

[0102] The specific steps of Comparative Example 1 included: thoroughly stirring the wastewater at 800 r / min using a magnetic stirrer, then adding sodium hypochlorite to achieve a concentration of 0.1 mg / L to initiate the reaction. During the process, 1 mL samples were taken at 0, 2, 4, 6, 8, and 10 min, and immediately quenched with sodium thiosulfate. The samples were then subjected to 10... 1 Up to 10 5 After serial dilution, take 0.1 mL and dilute 10 times. 5 The solution was spread onto agar plates containing tetracycline (16 mg / L), incubated at 37°C for 24 h, and the colony count and removal rate were calculated.

[0103] like Figure 2 As shown, in this comparative example, the removal of bacteria was approximately 0.51 log after 10 min.

[0104] Comparative Example 2

[0105] The method is an adjustment based on Comparative Example 1, the difference being that 0.5 mM hydrogen peroxide solution is added instead.

[0106] like Figure 2 As shown, in this comparative example, the removal of bacteria after 10 min was approximately 2.10 log.

[0107] Comparative Example 3

[0108] The method is an adjustment based on Example 1, the difference being that hydrogen peroxide solution is not added.

[0109] like Figure 2 As shown, in this comparative example, the removal of bacteria was approximately 1.10 log after 10 min.

[0110] like Figure 3 As shown, in this comparative example, the degradation capacity of the 19 resistance genes (ARGs) after 10 min was 0.453 log copy number mL. -1 .

[0111] like Figure 4 As shown, in this comparative example, the EPS content was measured to be as high as 9.01 mg / L.

[0112] like Figure 5 As shown in the comparative example, the specific energy consumption of this system is SEC = 1.51 kWh·m³. -3 ·log -1 The CT value required to degrade the resistance gene is 200 mg / min. -1 ·L -1 .

[0113] Comparative Example 4

[0114] The method is an adjustment based on Example 1, the difference being that sodium hypochlorite solution is not added.

[0115] like Figure 2 As shown, in this comparative example, the removal of bacteria was approximately 2.15 log after 10 minutes.

[0116] like Figure 3 As shown, in this comparative example, the degradation capacity of the 19 resistance genes (ARGs) was 0.600 log copy number mL after 10 min. -1 .

[0117] like Figure 4 As shown, in this comparative example, the EPS content was measured to be as high as 1.32 mg / L.

[0118] like Figure 5 As shown in the comparative example, the specific energy consumption of this system is SEC = 0.05 kWh·m³. -3 ·log -1 The CT value required to degrade the resistance gene is 1800 mg / min. -1 ·L -1 .

[0119] Comparative Example 5

[0120] The method is an adjustment based on Example 1, the difference being that sodium hypochlorite solution and hydrogen peroxide solution are not added.

[0121] like Figure 2 As shown, in this comparative example, the bacterial removal rate was approximately 0.10 log after 10 minutes.

[0122] like Figure 3As shown, in this comparative example, the degradation capacity of the 19 resistance genes (ARGs) was 0.0100 log copy number mL after 10 min. -1 .

[0123] like Figure 4 As shown, in this comparative example, the EPS content was measured to be as high as 0.50 mg / L.

[0124] Comparative Example 6

[0125] This is an adjustment based on Example 2, the difference being that potassium permanganate was not added and carbon felt material was not used.

[0126] like Figure 5 As shown, in this comparative example, the bacterial removal rate was 0.37 log after 15 min.

[0127] Comparative Example 7

[0128] The system is modified from Example 2, except that no electricity is applied and no potassium permanganate is added.

[0129] like Figure 6 As shown, in this comparative example, the bacterial removal rate was 0.07 log after 15 min.

[0130] Comparative Example 8

[0131] The system is modified from Example 2, but differs in that it is not powered and does not use carbon felt material.

[0132] like Figure 6 As shown, in this comparative example, the bacterial removal rate was 2.03 log after 15 min.

[0133] Comparative Example 9

[0134] This is an adjustment based on Example 2, the difference being that potassium permanganate was not added.

[0135] like Figure 6 As shown, in this comparative example, the bacterial removal rate was 0.23 log after 15 min.

[0136] Comparative Example 10

[0137] This is an adjustment based on Example 1, the difference being that carbon felt material was not used.

[0138] like Figure 6 As shown, in this comparative example, the bacterial removal rate was 2.44 log after 15 min.

[0139] Comparative Example 11

[0140] The system is modified from Example 2, except that it is not powered.

[0141] like Figure 6 As shown, in this comparative example, the bacterial removal rate was 1.98 log after 15 min.

[0142] III. Data Analysis

[0143] Figure 1 This study records the changes in spin-trapping EPR spectra at different reaction times during the EF / sodium hypochlorite / hydrogen peroxide reaction process in this application. The contribution of the active species to the inactivation of MDR *E. coli* and its resistance genes is investigated and analyzed. Figure 1 As shown, this demonstrates the existence of a two-stage synergistic combined oxidation system of "singlet oxygen non-radical oxidation disrupting cell wall - strong radical oxidation degradation gene". The first stage (0~2 min) uses singlet oxygen. 1 O2 is the dominant element, while the second stage (2-10 min) is dominated by free radicals (CRS). Singlet oxygen ( 1 O2 rapidly breaks down cell walls, causing cell membrane perforation within 2 minutes, exposing intracellular drug resistance genes to the oxidative system. Within 2-10 minutes, free radicals take over the oxidative degradation of the resistance genes.

[0144] Figure 2 These are the inactivation curves of antibiotic-resistant bacteria for different systems in Examples 1 and Comparative Examples 2-5 of this application, where EF represents electrolysis; NaClO represents sodium hypochlorite; H2O2 represents hydrogen peroxide; EF / NaClO represents electro-sodium hypochlorite; EF / H2O2 represents electro-hydrogen peroxide; and EF / NaClO / H2O2 represents electro-sodium hypochlorite-hydrogen peroxide. Figure 2It is evident that, under the same conditions, compared with the sodium hypochlorite system alone for inactivating antibiotic-resistant bacteria, this application improved the removal efficiency of antibiotic-resistant bacteria by 6.23 log, and compared with the hydrogen peroxide system alone for inactivating antibiotic-resistant bacteria, this application improved the removal efficiency of antibiotic-resistant bacteria by 5.18 log. This demonstrates that the effect of this application is significantly superior to other treatment methods, and is not simply a superposition of their effects. This is due to the complementary nature of their effects: singlet oxygen and other non-free radical species do not induce significant oxidative stress, but can selectively damage the bacterial cell membrane structure, causing membrane perforation, thereby exposing intracellular resistance genes to the oxidative system; while oxidative free radicals such as hydroxyl radicals can directly attack and degrade naked gene fragments, but if acted alone, they easily induce bacterial stress responses, leading to cell membrane thickening, hindering free radical entry, and resulting in ineffective drug consumption. By sequentially coupling sodium hypochlorite and hydrogen peroxide through electro-activation, the genes are first gently perforated and exposed using singlet oxygen, and then precisely oxidized and degraded by free radicals. The synergistic effect of the two processes avoids the shortcomings of a single process and significantly improves the removal efficiency of drug-resistant bacteria and genes.

[0145] Figure 3 The degradation performance of 19 resistance genes (ARGs) in different systems of Examples 1, 3, 4, and 5 of this application after 10 minutes of treatment is shown. Figure 3 As shown, the EF process alone resulted in negligible degradation of 19 ARGs within 10 minutes, indicating that direct electrode oxidation is insufficient to oxidize genetic fragments. Similarly, the EF / sodium hypochlorite and EF / hydrogen peroxide processes showed limited ARG degradation, ranging from 0.1 to 0.5 log copy number mL. -1 These results indicate that the oxidative effects of sodium hypochlorite and hydrogen peroxide, as well as the CRS and ROS that may be generated in the single oxidant 208 system, are insufficient to completely degrade ARG structures, possibly due to their limited oxidative strength or competitive consumption by cellular components. In contrast, the EF / sodium hypochlorite / hydrogen peroxide system achieved a higher ARG degradation rate than the EF / sodium hypochlorite and EF / hydrogen peroxide systems, with the average degradation rate significantly increasing from 0.453 and 0.600 to 2.79 log copy number mL. -1 This significant improvement indicates that the synergistic activation of sodium hypochlorite and hydrogen peroxide during electrofiltration produces a strong synergistic effect, rather than a simple additive contribution from individual oxidants. The observed synergistic effect may reflect changes in the composition, spatial distribution, and sequential dominance of the active substances, which requires further mechanistic investigation. Overall, these results demonstrate that this synergistic EF / sodium hypochlorite / hydrogen peroxide system not only achieves effective inactivation of MDR E. coli but also significantly degrades antibiotic resistance genes, highlighting its potential as a reliable disinfection strategy for controlling antibiotic resistance in water.

[0146] Figure 4 This figure represents the extracellular polymeric substances (EPS) secreted by multidrug-resistant *Escherichia coli* (MDRE. coli) in different systems of Examples 1, 3, 4, and 5 of this application (unit: mg / L). EPS is a self-protective substance secreted by bacteria when subjected to oxidative stress, and its content reflects the strength of the bacterial defense response. Figure 4 It can be seen that the EF / NaClO (chlorine radicals only) system produced the highest EPS content, at 9.01 mg / L. This indicates that chlorine radicals (CRS) have strong oxidizing properties (redox potential > 2 V), which strongly induces bacterial oxidative stress defense, leading to the secretion of large amounts of EPS to protect themselves. This is a major drawback of traditional chlorination disinfection. EF / NaClO / H2O2 (sequential oxidation: first...) 1 The EPS content in the CRS system after O2 exposure significantly decreased to 6.12 mg / L. This is because the initial singlet oxygen content decreased significantly. 1 O2 dominates the cell structure, with a low oxidation potential. It can damage cell structure but triggers almost no oxidative stress, exposing drug resistance genes to the system and thus "quietly" paving the way for subsequent attacks. Adding masking agents (NaHCO3 to scavenge chlorine free radicals, NaN3 to scavenge...) 1 When treated with O2 and chlorine free radicals, and with EF alone, EPS levels decreased to near background levels, ranging from 0.50 to 1.32 mg / L. This further validates... 1 O2 itself hardly triggers a defense response; chlorine free radicals are the main drivers of EPS secretion. Therefore, the two-stage synergistic oxidation system of this application, consisting of "singlet oxygen non-radical oxidation disrupting cell walls - strong free radical oxidative degradation of genes," is dominated by the sequential order of active species (from...) 1 O2 (from non-free radical to free radical) cleverly avoids the problem of bacteria secreting large amounts of EPS due to oxidative stress in traditional disinfection, creating conditions for chlorine free radicals to directly and efficiently attack resistance genes, and achieving cost-effective control of drug-resistant microorganisms and their genes.

[0147] Figure 5 This paper presents the CT values ​​and specific energy consumption assessment results of Examples 1, 3, and 4 of this application, as well as previously reported advanced disinfection processes, in the degradation of antibiotic resistance genes. Specific energy consumption (SEC) represents the electrical energy required to reduce the concentration of ARGs per unit volume by one logarithmic order, expressed in kilowatt-hours per cubic meter per logarithmic order. The CT value reflects the cumulative exposure to disinfectant required to achieve a specific inactivation level, expressed in milligrams per minute per liter, and is a standardized parameter for measuring oxidant utilization efficiency. Among the three comparative systems in this application, the core process EF / NaClO / H2O2 has the lowest SEC value, at 0.06 kW·h·m. -3·log -1 Furthermore, the CT value is extremely low, approximately 4–7 mg·min·L. -1 This achieves optimal energy consumption and oxidant utilization. In comparison, the SEC value of EF / NaClO is as high as 1.51 kW·h·m. -3 ·log -1 The CT value was 200 mg·min·L. -1 This indicates that using chlorine radicals alone not only results in high energy consumption but also significant waste of oxidant. While the EF / H2O2 SEC value is extremely low, at only 0.05 kW·h·m... -3 ·log -1 However, its CT value is as high as 1800 mg·min·L. -1 This means that it is almost impossible to effectively degrade resistance genes, and the oxidation capacity is severely insufficient. Therefore, only EF / NaClO / H2O2 achieves the lowest specific energy consumption and the highest oxidant utilization efficiency through a sequential oxidation mechanism, i.e., singlet oxygen first perforates and chlorine free radicals then degrade. The CT values ​​and SEC assessments of other reported advanced disinfection processes are detailed in Appendix Table 6.

[0148] Table 6. Specific energy consumption analysis of different disinfection methods for ARG damage

[0149]

[0150] Figure 6 These are the inactivation curves of resistant bacteria for different systems in Examples 2 and Comparative Examples 6-11 of this application, where C represents carbon felt alone; EC represents electro-carbon felt; E represents electrolysis; C-PM represents carbon felt-potassium permanganate; PM represents potassium permanganate; E-PM represents electro-potassium permanganate; and EC-PM represents electro-carbon felt-potassium permanganate. Figure 6 It was found that after 15 min of reaction, the logarithmic removal rate of AR E. coli in system C was 0.07 log, indicating that the adsorption and removal effect of carbon felt on E. coli was very weak. The logarithmic removal rates of ARE. coli in systems E and EC were 0.37 log and 0.23 log, respectively, indicating that the direct electron transfer in this application had almost no removal effect on E. coli. This may be due to the current density of 12.55 mA·cm⁻¹ used in the experiment. -2The current (0.15 A) was relatively low, and the anode could not generate enough in-situ oxides, making it impossible to effectively inactivate AR E. coli in a short time. The logarithmic removal rates of PM, C-PM, and E-PM for AR E. coli were 2.03 log, 1.98 log, and 2.44 log, respectively. The results indicate that E alone activated PM, which had a weak inactivation ability for AR E. coli, and C alone could not activate PM. However, the logarithmic removal rate of the EC-PM system reached 4.92 log, which was significantly enhanced compared to PM alone, with a logarithmic removal rate increase of 2.89 log. This suggests that when carbon felt is used as the cathode, there is a very significant synergistic effect between electrophoresis and potassium permanganate.

[0151] Figure 7 This document presents the degradation kinetics curves of pollutants in the EF / PM system after the addition of different active species quenchers in Example 2 of this application. Figure 7 It is evident that the degradation degree of the EF / PM system without quenchers was -5.0 at 15 min, indicating efficient removal of pollutants. With the addition of free radical quenchers MeOH and TBA, the degradation degrees at 15 min were -4.2 and -3.2, respectively, which were weaker than the system without quenchers (-5.0), suggesting that free radicals (such as ·OH and SO4·⁻) contribute to this system. The addition of L-His (¹O2 quencher) resulted in a degradation degree of -4.8, close to the system without quenchers, indicating only slight inhibition; however, the addition of PP (Mn(III) complexing agent) caused a sharp decrease in degradation degree to -1.4, demonstrating the most significant inhibition effect. These results indicate that Mn(III) in the EF / PM system... a q is the dominant active species, while the contributions of ¹O2 and free radicals are relatively small. Overall, the non-radical pathway in this system mainly originates from the soluble Mn(III) generated by the electroreduction activation of PM.

[0152] Figure 8 These are the UV-Vis absorption spectra of the EF / PM system at different time points during the reaction process in Example 2 of this application. Figure 8 It can be seen that as the reaction time increases, the characteristic absorption peak of Mn(VII) near 525 nm gradually weakens, indicating that potassium permanganate is continuously consumed; at the same time, the characteristic absorption peak of Mn(III) gradually appears at about 460 nm. aq A new absorption peak appears, and the intensity of this peak increases with increasing reaction time. The spectrum at 15 min of reaction is similar to that of pure Mn(III). aq The standard spectra were largely consistent. Furthermore, the PMSO probe experiment detected the formation of PMSO2, further confirming the presence of high-valent manganese species (Mn(III)). aqThe presence of Mn(III) leads to the in-situ generation of Mn(III) through PM activation via electrofiltration in the EF / PM system. aq This enables the efficient degradation of organic pollutants.

[0153] Figure 9 This is an electrochemical reactor and schematic diagram of Example 3 of this application. Figure 10 The figures show the EPR spectra of the EF / PMS system in Example 3 of this application under different spin trapping agents (DMPO and TEMP) during the reaction process. As shown in the figure, when TEMP is used as the trapping agent, a triplet characteristic peak with an intensity ratio of approximately 1:1:1 appears in the EPR spectrum. This is a typical signal of singlet oxygen (¹O2), indicating that ¹O2 is generated in the system. In contrast, when DMPO is used as the trapping agent, no characteristic adduct signal of the corresponding free radical (such as ·OH or SO4·⁻) (e.g., a 1:2:2:1 quartet of ·OH) was detected, further confirming that non-radical pathways, especially singlet oxygen (¹O2), are the main active species in this EF / PMS system.

[0154] Figure 11 The figures show the degradation kinetics curves of pollutants in the EF / PMS system after adding different active species quenchers in Example 3 of this application. The results are as follows... Figure 11 As shown. By Figure 11 It is evident that the addition of L-histidine (¹O2-specific quencher) significantly inhibited pollutant degradation, with a reaction degree of only -0.5 at 15 min; while the reaction degree of the EF / PMS system without quencher was -1.0 at 15 min. The addition of free radical quenchers MeOH, TBA, and DMSO resulted in reaction degrees of -1.1, -1.2, and -1.3 at 15 min, respectively. Compared to the system without quencher (-1.0), no significant inhibition was observed (DMSO even slightly enhanced the reaction degree, possibly due to experimental fluctuations or the additional effect of DMSO). These results indicate that ¹O2 is the dominant reactive species in the EF / PMS system, while free radicals (such as ·OH and SO4·⁻) contribute relatively little.

[0155] IV. Summary

[0156] (1) Examples 1 to 5 respectively constructed time-controlled two-stage reaction pathways based on different oxidants and electro-activation systems. By comparing with comparative examples using only a single oxidant, single electro-activation, or no active species with time-distinguishing features, it was confirmed that the time-controlled oxidation system of this application can significantly improve the inactivation efficiency of drug-resistant bacteria and the degradation depth of intracellular antibiotic resistance genes. Among them, the EF / NaClO / H2O2 system constructed in Example 1 can achieve a logarithmic removal rate of 7.33 log for drug-resistant bacteria within 10 minutes, and the average log removal value of 19 drug resistance genes is 2.79 log copies / mL. The treatment effect is significantly better than the single electro-activated sodium hypochlorite system and the single electro-activated hydrogen peroxide system in the comparative examples. Moreover, the final efficiency is much higher than the simple superposition of the effects of the two single systems, directly verifying the synergistic effect of the two-stage pathway. From the efficiency level, it solves the core technical bottleneck of existing single oxidation technologies that are difficult to break through bacterial biological barriers and cannot efficiently and thoroughly degrade intracellular drug resistance genes.

[0157] (2) Meanwhile, by comparing the content of extracellular polymers in different systems, the amount of bacterial extracellular polymer secretion in the system of Example 1 was reduced by about 32% compared with the single chlorine radical system. Combined with the quenching experiment results, it can be seen that the non-radical oxidation process dominated by singlet oxygen in the first stage can avoid the oxidative stress response caused by strong free radicals contacting the bacterial cells in advance, and reduce the secretion of protective substances such as extracellular polymers. This low-stress characteristic has a close positive synergistic effect with the subsequent efficient gene degradation effect. The reduction of extracellular polymer secretion not only eliminates the physical protective layer on the surface of the bacterial cells, allowing non-radical species to act on the cell membrane more smoothly to complete the cell wall breaking, but also reduces the total amount of reducing interference substances in the system, reduces the ineffective quenching loss of free radicals in the second stage, and allows free radicals to act more concentratedly on the exposed drug resistance gene fragments, further amplifying the gene degradation efficiency and forming a positive cycle of "low stress - easy cell wall breaking - high targeting - strong degradation".

[0158] (3) Regarding adaptability to complex water quality, the data from the examples confirm that the two-stage system of this application can still maintain stable performance in inactivating drug-resistant bacteria and degrading genes in environments containing typical interfering matrices such as high concentrations of dissolved organic matter, bicarbonate, and chloride ions. It can achieve a drug-resistant bacteria inactivation rate greater than 6.56 log in a wide pH range of 3 to 11, and the drug-resistant gene removal rate remains above 99%. In contrast, the traditional free radical oxidation system in the comparative example shows a significant decrease in treatment efficiency under the same interference conditions. This strong anti-interference performance and the aforementioned high-efficiency degradation effect form a mutually supportive relationship. The non-free radical pathway in the first stage itself has low sensitivity to the background matrix of the water body and is not easily quenched, ensuring the stability of the cell wall breaking process under complex water quality. The stable and uniform cell wall breaking effect provides a continuous and controllable reaction substrate for the free radical degradation in the second stage, avoiding fluctuations in degradation efficiency caused by incomplete cell wall breaking. This allows the system to maintain high-efficiency treatment performance under complex water quality conditions in actual water bodies, solving the technical problems of poor resistance to water quality fluctuations and unstable treatment effects in practical applications of traditional advanced oxidation processes.

[0159] (4) In terms of operational efficiency and environmental safety, the energy consumption and CT value analysis of the embodiments show that the specific energy consumption for the system of this application to achieve 1-log degradation of drug resistance genes is only 0.06 kWh·m³. -3 ·log -1 The required CT value is significantly lower than that of similar advanced oxidation processes based on sodium hypochlorite and hydrogen peroxide, and is significantly better than the comparative systems. This energy efficiency improvement is directly related to the aforementioned high selectivity and low loss characteristics. The sequential pathway design of this application allows non-radicals and free radicals to target their respective functions at the corresponding stages, reducing ineffective reactions with the background matrix and oxidant loss caused by bacterial defense. While improving the treatment effect, it simultaneously reduces the total amount of oxidant added and the power consumption. The reduction in the amount of oxidant added further reduces the potential risk of halogenated disinfection byproducts generated by the reaction of chlorine-based oxidants with organic matter in the water. It takes into account treatment efficiency, operating cost and environmental friendliness, and solves the problem of high cost and secondary pollution risk caused by excessive addition of oxidant in traditional processes in pursuit of treatment effect.

[0160] The documents referenced in Tables 2 and 6 of this application are as follows:

[0161] Reference 1: Sun, W.; Xie, M.; Shi, B.; Zhu, Z.; Zhang, J.; Wang, Y.; Bian, J.; Wang, H.; Zhao, C. Electrofiltration-activated peroxymonosulfate and permanganate processes for nonradical oxidation of antibiotic-resistant contaminants: comparing 1 O2 with active Mn(III)aq. Chem. Eng. J. 2025, 524, 169230. https: / / doi.org / 10.1016 / j.cej.2025.169230.

[0162] Reference 2: Ni, X.; Hou, X.; Ma, D.; Li, Q.; Li, L.; Gao, B.; Wang, Y. Simultaneous removal of antibiotics and antibiotic-resistant genes using a CeO2@CNT electrochemical membrane-NaClO system. Chemosphere 2023, 338, 139457. https: / / doi.org / 10.1016 / j.chemosphere.2023.139457. <0OO0558>Reference 3: Zheng, Q.; Zhang, Y.; Qianxin; Zhang; Wang, Y.; Yu, G. Removal of antibiotic-resistant bacteria and plasmid-encoded antibiotic resistance genes in water by ozonation and electro-peroxone process. Chemosphere 2023, 319, 138039. https: / / doi.org / 10.1016 / j.chemosphere.2023.13,8039.

[0164] Literature 4: He, H.; Zhao, T.; Ma, Q.; Yang, X.; Yue, Q.; Huang, B.; Pan, X. Photoelectrocatalytic coupling system synergistically removal of antibiotics and antibiotic-resistant bacteria from aquatic environment. ‌J. Hazard. Mater. 2022, 424, 127553. https: / / doi.org / 10.1016 / j.jhazmat.2021.127553.

[0165] Literature 5: Li, Z.; Lv, H.; Tong, K.; He, Y.; Zhai, C.; Yun, Y.; Zhu, M. Modulating the precursors of carbon nitride to boost local electron delocalization for H2O2 photosynthesis to remove oxytetracycline and its antibiotic-resistant genes. Appl. Catal. B Environ. 2024, 345, 123690. https: / / doi.org / 10.1016 / j.apcatb.2024.123690.

[0166] Literature 6: Liu, Q.; Chen, Y.; Mei, T.; Wang, S.; Zhang, L.; Sun, H.; Liao, X. Oxalated zero-valent iron catalyzed ozonation for effective removal of antibiotic resistance genes from municipal sewage sludge. J. Environ. Chem. Eng. 2023, 11 (3), 109717. https: / / doi.org / 10.1016 / j.jece.2023.109717.

[0167] Literature 7: Gao, B.; Dou, M.; Wang, J.; Li, S.; Wang, D.; Ci, L.; Fu, Y. Efficient persulfate activation by carbon defects g-C3N4 containing electron traps for the removal of antibiotics, resistant bacteria and genes. Chem. Eng. J. 2021, 426, 131677.

[0168] Literature 8: Li, H.; Lu, L.; Yin, W.; Liu, H. Integration of Nanowire-confined electroporation of antibiotic-resistant bacteria and electroactivation of peracetic acid for eliminating intracellular resistance genes. J. Jazard. Mater.‌ 2025, 497, 139752. https: / / doi.org / 10.1016 / j.jhazmat.2025.139752.

[0169] Literature 9: Ma, H.; Zhang, L.; Huang, X.; Ding, W.; Jin, H.; Li, Z.; Cheng, S.; Zheng, L. A novel three-dimensional galvanic cell enhanced Fe 2+ / persulfate system: High efficiency, mechanism and damaging effect of antibiotic-resistant E. coli and genes. Chem. Eng. J. 2019, 362, 667–678. https: / / doi.org / 10.1016 / j.cej.2019.01.042.

[0170] Reference 10: Qu, C.; Soomro, G. S.; Ren, N.; Liang, D.; Lu, S.; Xiang, Y.; Zhang, S. Enhanced electro-oxidation / peroxone (in situ) process with a Ti-based nickel-antimony doped tin oxide anode for phenol degradation. ‌J. Hazard. Mater.‌ 2020, 384, 121398. https: / / doi.org / 10.1016 / j.jhazmat.2019.121398.

[0171] Reference 11: Lu, X.; Zhou, X.; Qiu, W.; Wang, Z.; Cheng, H.; Zhang, H.; Yu, J.; Wang, D.; Ma, J. Singlet oxygen involved electrochemical disinfection by anodic oxidation of H2O2 in the Presence of Cl − . Chem. Eng. J. 2022, 446, 136871. https: / / doi.org / 10.1016 / j.cej.2022.136871.

[0172] Reference 12: Guo, D.; Liu, Y. Singlet oxygen-mediated electrochemical filter for selective and rapid degradation of organic compounds. Ind. Eng. Chem. Res. 2020, 59 (31), 14180–14187. https: / / doi.org / 10.1021 / acs.iecr.0c02223.

[0173] Document 13: Ren, N.; Qu, C.; Zhang, A.; Yu, C.; Li, (51), 22829–22839. https: / / doi.org / 10.1021 / acs.est.4c09855.

[0174] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and not to limit the technical solutions. Those skilled in the art should understand that any modifications or equivalent substitutions to the technical solutions of this application without departing from the spirit and scope of this technical solution should be covered within the scope of the claims of this application.

Claims

1. A method for rapidly blocking the spread of antibiotic resistance genes in water, characterized in that, The method is carried out in the following stages: In the first stage, wastewater containing antibiotic-resistant bacteria and their resistance genes is treated to form non-free radical species in the wastewater. These non-free radical species are then used to oxidize and disrupt the cell membrane structure of the antibiotic-resistant bacteria, exposing their internal resistance genes to the oxidation system. The non-free radical species are singlet oxygen or Mn(III). In the second stage, a free radical-dominated oxidation system reaction is generated in the wastewater treated in the first stage to break down and mineralize the exposed drug resistance genes, thereby blocking their spread in the aquatic environment. In the second stage, the free radicals are selected from one or more of the following: hydroxyl radicals •OH, superoxide radicals O2. •− iodate radical IO3 • Chlorine-active species.

2. The method of claim 1, wherein, In the first stage, wastewater containing antibiotic-resistant bacteria and their resistance genes is treated to generate singlet oxygen in the wastewater. 1 O2 is maintained for 1 to 5 minutes, and singlet oxygen in the wastewater is maintained. 1 The steady-state concentration of O2 remains at 1×10⁻⁶. -10 mol / L or higher; or generate Mn(III) to maintain the steady-state concentration of Mn(III) at 10⁻ 7 mol / L~10⁻ 5 mol / L; meanwhile, in the first stage, the steady-state concentration of each free radical remained at 1×10 mol / L. - 13 Below mol / L.

3. The method of claim 1, wherein, In the second stage, the reaction solution obtained after the first stage is further processed to allow the free radicals generated in the reaction solution to continue processing for 5 to 15 minutes; during this process, the steady-state concentration of each free radical is maintained at 1 × 10⁻⁶. -12 Above mol / L.

4. The method according to claim 2 or 3, characterized in that, The chlorine-active species CRS are Cl•, ClO• and Cl2•. - One or more of them.

5. The method of claim 1, wherein, The first and second stages are implemented using the following electrochemical methods to promote the generation of singlet oxygen and free radicals: (1) Add an electrolyte to the wastewater. The electrolyte is selected from one or more of hydrochloride or sulfate, and the electrolyte is selected from at least one of NaCl, KCl, Na2SO4, and K2SO4. The concentration of the electrolyte is 40 mmol / L to 150 mmol / L. (2) The current density is controlled at 2~30 mA / cm. 2 The optimal current density is 12.55 mA / cm². 2 ; (3) First stage: Add the first oxidant, which is selected from at least one of hydrogen peroxide, sodium hypochlorite, monosodium persulfate, and potassium permanganate; the dosage of the first oxidant in the wastewater is 0.01 g / L to 1 g / L; adjust the pH of the reaction system to 7 to 9; (4) Second stage: Add a second oxidant, which is selected from peracetic acid or periodate; the dosage of the second oxidant in the wastewater is 0.01 g / L to 1 g / L.

6. The method of claim 1, wherein, The wastewater also includes at least one interfering substance selected from at least one of dissolved organic matter, humic acid, bicarbonate ions, chloride ions, and hydrogen phosphate ions; and in the presence of the interfering substance, the method has an inactivation rate of >6.56 log for antibiotic-resistant bacteria.

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